JPH0425253B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a preventive agent for carcinogenesis such as liver cancer and gastric cancer derived from an N-nitroso compound.

It is well known today that various N-nitroso compounds, typically N-nitrosodimethylamine (NDMA), have strong carcinogenic effects even in trace amounts (H. Druckrey et al., Z. Krebsforsh, 69 ).
103 (1967); JHHotchkiss et al., J.Assoc.Off.
Anal.Chem., 64 1037 (1981) and GGGibson
et al., “Safety Evaluation of Nitrostable
Drug and Chemicals “Tayler and Francis
Ltd., London, 1981, etc.). Furthermore, N-nitrosamines such as NDMA, which have a particularly strong carcinogenic potential, are converted into nitrites in the body from nitrates, which are abundant in foods such as vegetables, and aliphatic amines, which are also found in fish meat. It is also becoming increasingly clear that it is a carcinogenic factor that is produced by reactions in body organs such as the stomach (Correa, P.
et al., Lancet II, 58-60 (1975);
Tannenbaum, SR et al., Annals of New
York Academy of Sciences 355 267−277
(1980); Tannenbaum, S.R. et al., Cancer
Lett. 14 131-136 (1981); DHFine et al.
Nature 265 753 (1977) and P. Schlag et al.
Lancet, April, 5 727 (1980), etc.).

In view of the above, the present inventors have conducted extensive research into substances that effectively suppress or inhibit the in vivo production of carcinogens and detoxify their precursors, and have found that proline, an amino acid, is present in the stomach. It reacts markedly and preferentially with nitrite ions to give nontoxic N-nitrosoproline (NPRO) when coexisting with aliphatic amines under these conditions, resulting in the in vivo formation of carcinogenic N-nitroso compounds. The present invention was achieved based on the finding that the reaction mixture can be inhibited very effectively and the entire reaction mixture can be rendered substantially harmless even at the bioassay level.

The active ingredients, pharmacological and biological effects, dosage and administration, toxicity, etc. of the agent of the present invention will be explained in detail below.

Active Ingredients The active ingredients of the present agent include naturally occurring L-proline, protein hydrolysates, synthetic DL-proline, and its hydrate form (C ₁₀ H ₁₈ N ₂ O ₄・H ₂ O ), and various salts thereof such as sodium salts and potassium salts can be suitably used.

Pharmacological or Biological Effects As shown in the experimental examples below, pre-added proline has the effect of virtually completely detoxifying the carcinogenicity of secondary amines and nitrite-containing solutions, and therefore N-
It can be said that it is extremely useful as a carcinogenic preventive drug derived from a nitroso compound from the viewpoint of preventive medicine. Here, the amount of proline required for complete detoxification is approximately equimolar or more to the amount of nitrite.

It has already been reported that the generated NPRO is not carcinogenic and is almost completely and rapidly excreted in the urine (Chu, C., & Magee,
PNCancer Res., 41 3653-3657 (1981);
Dailey, REet al., Toxicol., 3 23-28
(1975); Mirvish, SSet al., J.Natl.Inst. 64
1435-1442 (1980) and Ohshima, H & Bartsch,
H. Cancer Res., 41 3658-3662 (1981), etc.).

Usage/Administration Nitrate ingested from foods such as vegetables is converted to nitrite by microorganisms in the oral cavity, etc., and reacts with amines, which are components of the ingested food, mainly in the stomach to produce carcinogenic substances. has been done. For Japanese people, the amount of nitrate intake from food is 218 to 408
mg/day, and the concentration of nitrite ions in saliva is about several ppm to 50 ppm (Ishiwata,
H. et al., Proceedings of the 3rd
International Conference on Environmental
Mutagens, Tokyo Sept.21-27, 1981 page571
-575), and the concentration of nitrite in gastric fluid is 0.1 to several tens of μ
g/ml (P. Schlag et al., Lancet, April 5
727 (1980), the dose of the present agent is several 100 μg to several g/g of proline.
50Kg body weight per day, more preferably 1mg to 1g/day.
It is within the range of 50 kg body weight, and from the viewpoint of preventive medicine, 50 to 500 mg/day/50 kg body weight can actually have a sufficient cancer prevention effect.

Incidentally, the lowest carcinogenic amount of NDMA is estimated to be approximately 1 ppm, which is equivalent to 50 mg based on a human body weight of 50 kg (M. Aral et al., Gann 70 549 (1979),
etc).

On the other hand, although the agent of the present invention is generally administered orally, it may also take a dosage form in which it is preferably added to and ingested with food. In addition, its dosage forms include aqueous solutions, powders, granules, tablets, capsules,
Sustained-release microcapsules and the like can be appropriately selected and implemented by preparing a conventional dosage form alone with or without an appropriate carrier such as purified starch powder, filler, or diluent.

The agent of the present invention can also be naturally ingested by being added or blended in advance to foods such as the aforementioned foods with high secondary amines and nitrate content, as well as seasonings such as soybean oil, sauces, and mayonnaise. Since it can also take the form of a drug, it can exhibit even more preventive effects.

On the other hand, since the natural amino acid L-proline is abundantly contained in natural products such as gelatin, the hydrolyzed products of these may be used as they are as the agent of the present invention.

Toxicity It goes without saying that proline and its salts are food ingredients, so they are completely non-toxic orally unless they are ingested excessively.

Incidentally, DL-proline was added to ICR mice (male 6
Samples of 1 mg, 10 mg, and 100 mg (dissolved in 0.5 ml of physiological saline) were administered intraperitoneally to mice (week old, average weight 35.0 ± 0.3 g, 10 animals in each group), and their survival was observed for 14 days. However, the LD ₅₀ value calculated according to the Behrens-Ka¨rber method was 760 mg/Kg body weight (mouse) or higher.

Experimental example 1 In vitro experiment reaction solution composition by Ames test The reaction PH of dimethylnitrosamine production reaction between dimethylamine (hydrochloride) and sodium nitrite
Generation rate of 15% in 3.4 [Mirvish.SS, J.Nat.
cancer Inst., 44 633 (1970)].
The reaction amount was set as follows so that the product was 20 mg in 1 ml of the reaction solution.

Dimethylnitrosamine production reaction solution Dimethylamine hydrochloride: A solution containing 92 mg/ml in terms of dimethylamine was prepared, and 0.75 ml of it was used. Sodium nitrite...A 142 mg/ml solution was prepared, and 0.75 ml of it was used.

The pH of the two above was adjusted to 3.4 with conc HCl, and an appropriate amount of distilled water was added to prepare a 3 ml reaction solution.

1 ml of each of proline addition reaction solutions dimethylamine hydrochloride and sodium nitrite having the above formulations was used.

L-proline...Prepare a 238 mg/ml solution, and add 0.95 ml of it to 1/2 molar equivalent to nitrite.
1.9 ml was used as 1 molar equivalent.

When adding proline, mix dimethylamine and proline, then add sodium nitrite.
The pH was adjusted to 3.4 with conc HCl and an appropriate amount of distilled water was added to make a 4 ml reaction solution.

Reaction Conditions Each reaction solution was sealed tightly, covered with aluminum foil to shield it from light, and the conditions in the stomach were set, and the reaction was carried out by incubating at 37°C for 3 hours.

Mutagenicity test method In order to examine the increase in mutagen depending on the amount of reaction solution, samples of each reaction solution were set to 25, 50, 75, and 100μ to determine the linearity of the reaction solution. Also,
The amount of S-9 is 150μ/500μmix, and Pre
Incubation was performed at 25°C for 40 minutes, and TA-100 was used as the bacterial strain.

[Yazukuri. , Mutation Research., 48 , 121-130
(1977). ] The results are shown in Figures 1 and 2.

In Figure 1, the horizontal axis is the amount of dimethylnitrosamine-forming reaction liquid (μ), and the vertical axis is the return (His ⁺ ).
This is the number of colonies. As is clear from the figure, since linearity was achieved when the reaction solution volume was 50μ or less, this was set as the dose range in this test.

On the other hand, in Fig. 2, both axes are the same as above, but
Curve A is data for a reaction solution without the addition of proline, curve B is data for a reaction solution containing 1/2 molar equivalent of proline, and curve C is data for a reaction solution containing 1 molar equivalent of proline.

As is clear from FIG. 2, the addition of 1/2 molar equivalent of proline has a sufficient mutagenicity suppressing effect. It has also been separately confirmed that NPRO is not mutagenic.

Experimental example 2.
Vivo experiment It was reported that in response to DNA damage caused by carcinogen administration, the amount of NAD in organs decreased, showing organ specificity. [Sakamoto et al. , Japanese Mutagen Society, 1981] Therefore, we conducted the following experiment focusing on the liver specificity of dimethylnitrosamine.

Test Animals Three male Wistar rats (5 weeks old) were included in each group, and in each test, one group served as an untreated control group.

Administered substances and administration method Reaction solutions similar to those used in the Ames test were prepared and administered orally using a probe at a dose of 0.1 ml per 100 g of animals.

Each reaction solution administration group was sacrificed by decapitation after a certain period of time, and after sufficient bloodletting, the abdomen was immediately opened to remove the liver. Approximately 1 g of liver per animal was accurately weighed to make 4 specimens. I tried to minimize it as much as possible. Specimens were immediately frozen.

Methods of Enzymatic Analysis., Vol 4,
2045 (1974) using ethanol as a substrate.
ADH (alcohol dehydrogenase, 1.2mg/
The measurement was carried out by an enzymatic method using ml). That is, after weighing, the frozen specimen is homogenized in a frozen state, and 5 ml of 0.6N-HClO ₄ is added thereto to remove protein. The sample was centrifuged at 3000 rpm for 5 minutes to obtain a supernatant, and 0.2 ml of 1M-K ₂ HPO ₄ was added per 1 ml of supernatant, and the pH was adjusted to 7.0 with 3N-KOH.
Adjust to neutrality of 7.4. This was further centrifuged at 3000 rpm for 5 minutes, and 1 ml of the supernatant after removing the KClO ₄ precipitate was collected.
Add 1 ml of 0.1M sodium pyrophosphate/semicarbazide hydrochloride buffer (PH8.8),
After adding 10μ of ethanol and stirring, the OD at 340nm is measured (E ₁ ). Add 10μ of ADH
In addition, OD was measured in the same manner (E ₂ ), the difference between E ₂ −E ₁ (ΔE _340on ) was calculated, and 1 g of liver was calculated using a known calculation method.
The amount of NAD (μmole) per sample was determined.

Experimental results Changes due to oral administration of dimethylnitrosamine The changes in the amount of NAD in the liver were investigated using commercially available dimethylnitrosamine at doses of 25mg/Kg and 100mg/Kg.

The measurement time was 5, 10, 24 according to the report of Sakamoto et al.
It was an hour later. The results are shown in FIG. In the figure, the vertical axis is the amount of NAD in the liver (μmole/g), and the horizontal axis is time (hr) (the same applies to Figures 4 and 5 below).

Changes due to administration of each reaction solution Take 0.5 ml each of dimethylamine hydrochloride and sodium nitrite solutions at the above concentrations, add 0.25 to 2 molar equivalents of L-proline solution to sodium nitrite, and react in the same manner as above. A solution was prepared and administered to rats, and the liver NAD amount (μmole/g) was measured over time.

The results are shown in Figure 4. In the figure, curves A, B, C
and D are respectively no proline added, 1 equivalent added, and 2
Corresponds to equal addition and control levels, respectively.

In addition, administration of an amount equivalent to 2 equivalents of proline alone (FIG. 5A) and administration of an amount equivalent to nitrosoproline production alone (FIG. 5B) was also investigated.

Next, change the amount of proline added to determine the amount of NAD at 10 hours after administration, when the amount of NAD decrease is maximum.
A dose-response curve (Figure 6A) was determined. In the figure, the vertical axis is the amount of NAD, the horizontal axis is the molar equivalent,
Curve B shows the control level.

It was also separately confirmed that a decrease in the amount of NAD in the liver was highly correlated with an increase in unscheduled DNA synthesis, which is a more direct indicator of carcinogenicity.

[Brief explanation of drawings]

The attached FIGS. 1 to 6 are explanatory diagrams of experimental examples of the present invention.

Claims (1)

[Claims] 1. A carcinogenic preventive agent derived from an N-nitroso compound, characterized by containing proline and/or a salt thereof as an active ingredient.